In the small intestine, stem cells are located deep in the crypts of Leiberkiihn and have been demonstrated to be multipotent precursors of the 4 principal lineages within the epithelium. Understanding the biology of these stem cells is central to the development of effective rejuvenative therapies for intestinal failure and short gut syndrome. Moreover, in situations where the stem cells themselves are damaged, transplantation of healthy stem cells may afford a novel and effective therapy. Although the in vivo behavior (e.g. kinetics) of intestinal stem cells has been studied for many years, our understanding of the properties of these cells has been greatly hampered by lack of methods to isolate, identify and characterize them. In work funded by an R21 grant we have used a flow cytometry method to obtain a side population (SP) of viable cells from mouse small intestine which are greatly enriched in the intestinal stem cell marker Musashi-1. With this evidence, we believe that our SP includes the epithelial stem cells and that SP cells are worthy of further study to elucidate their basic biology and to explore their therapeutic potential. Thus, the Specific Aims of this proposal are: 1) To transplant small intestinal SP cells into damaged regions of mouse small intestine and subsequently assess the morphology as well as the expression of lineage-specific markers in epithelium derived from the transplanted cells. 2) To perform microarray analysis on RNA from small intestinal SP cells as compared with non-SP cells in order to identify mRNAs that are enriched in the SP. Via the Stem Cell Genome Anatomy Project these data will be compared with those from a putative intestinal stem cell fraction obtained by a different approach. 3) To identify additional markers for intestinal epithelial stem cells by performing high throughput in situ hybridization on adult and infant small intestine using probes to known markers and transcripts of stem cells from other tissues. Although these represent an ambitious set of aims we believe they are wothy because they will set the stage for a variety of future investigations regarding both the basic biology and the therapeutic potential of intestinal epithelial stem cells.